1. Field of the Invention
The present invention relates generally to the fields of molecular biology, developmental biology, cancer biology and medical therapeutics. Specifically, the present invention relates to novel de novo DNA cytosine methyltransferases. More specifically, isolated nucleic acid molecules are provided encoding mouse Dnmt3a, and Dnmt3b and human DNMT3A and DNMT3B de novo DNA cytosine methyltransferase genes. Dnmt3a and Dnmt3b mouse and DNMT3A and DNMT3B human polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are isolated nucleic acid molecules encoding mouse Dnmt3a2 and human DNMT3A2, which are small forms of the corresponding Dnmt3a mouse and DNMT3A human genes. Dnmt3a2 mouse and DNMT3A2 human polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to an in vitro method for cytosine C5 methylation. Also provided is a diagnostic method for neoplastic disorders, and methods of gene therapy using the polynucleotides of the invention.
2. Related Art
Methylation at the C-5 position of cytosine predominantly in CpG dinucleotides is the major form of DNA modification in vertebrate and invertebrate animals, plants, and fungi. Two distinctive enzymatic activities have been shown to be present in these organisms. The de novo DNA cytosine methyltransferase, whose expression is tightly regulated in development, methylates unmodified CpG sites to establish tissue or gene-specific methylation patterns. The maintenance methyltransferase transfers a methyl group to cytosine in hemi-methylated CpG sites in newly replicated DNA, thus functioning to maintain clonal inheritance of the existing methylation patterns.
De novo methylation of genomic DNA is a developmentally regulated process (Jahaner, D. and Jaenish, R., “DNA Methylation in Early Mammalian Development,” In DNA Methylation: Biochemistry and Biological Significance, Razin, A. et al., eds., Springer-Verlag (1984) pp. 189-219 and Razin, A., and Cedar, H., “DNA Methylation and Embryogenesis,” in DNA Methylation: Molecular Biology and Biological Significance, Jost., J. P. et al., eds., Birkhäuser Verlag, Basel, Switzerland (1993) pp. 343-357). It plays a pivotal role in the establishment of parental-specific methylation patterns of imprinted genes (Chaillet, J. R. et al., Cell 66:77-83 (1991); Stöger, R. et al., Cell 73:61-71 (1993); Brandeis, M. et al., EMBO J. 12:3669-3677 (1993); Tremblay, K. D. et al., Nature Genet. 9:407-413 (1995); and Tucker, K. L. et al., Genes Dev. 10:1008-1020 (1996)), and in the regulation of X chromosome inactivation in mammals (Brockdoff, N. “Convergent Themes in X Chromosome Inactivation and Autosomal Imprinting,” in Genomic Imprinting: Frontiers in Molecular Biology, Reik, W. and Sorani, A. eds., IRL Press Oxford (1997) pp. 191-210; Ariel, M. et al., Nature Genet. 9:312-315 (1995); and Zucotti, M. and Monk, M. Nature Genet. 9:316-320 (1995)).
Thus, C5 methylation is a tightly regulated biological process important in the control of gene regulation. Additionally, aberrant de novo methylation can lead to undesirable consequences. For example, de novo methylation of growth regulatory genes in somatic tissues is associated with tumorigenesis in humans (Laird, P. W. and Jaenisch, R. Ann. Rev. Genet. 30:441-464 (1996); Baylin, S. B. et al., Adv. Cancer. Res. 72:141-196 (1998); and Jones, P. A. and Gonzalgo, M. L. Proc. Natl. Acad. Sci. USA 94:2103-2105 (1997)).
The gene encoding the major maintenance methyltransferase, Dnmt1, was first cloned in mice (Bestor, T. H. et al., J. Mol. Biol. 203:971-983 (1988), and the homologous genes were subsequently cloned from a number of organisms, including Arabidoposis, sea urchin, chick, and human. Dnmt1 is expressed ubiquitously in human and mouse tissues. Targeted disruption of Dnmt1 results in a genome-wide loss of cytosine methylation and embryonic lethality (Li et al., 1992). Interestingly, Dnmt1 is dispensable for the survival and growth of the embryonic stem cells, but appears to be required for the proliferation of differentiated somatic cells (Lei et al., 1996). Although it has been shown that the enzyme encoded by Dnmt1 can methylate DNA de novo in vitro (Bestor, 1992), there is no evidence that Dnmt1 is directly involved in de novo methylation in normal development. Dnmt1 appears to function primarily as a maintenance methyltransferase because of its strong preference for hemi-methylated DNA and direct association with newly replicated DNA (Leonhardt, H. et al., Cell 71:865-873 (1992)). Additionally, ES cells homozygous for a null mutation of Dnmt1 can methylate newly integrated retroviral DNA, suggesting that Dnmt1 is not required for de novo methylation and an independently encoded de novo DNA cytosine methyltransferase is present in mammalian cells (Lei et al., 1996).
Various methods of disrupting Dnmt1 protein activity are known to those skilled in the art. For example, see PCT Publication No. WO92/06985, wherein mechanism based inhibitors are discussed. Applications involving antisense technology are also known; U.S. Pat. No. 5,578,716 discloses the use of antisense oligonucleotides to inhibit Dnmt1 activity, and Szyf et al., J. Biol. Chem. 267: 12831-12836, 1992, demonstrates that myogenic differentiation can be affected through the antisense inhibition of Dnmt1 protein activity.
Thus, while there is a significant amount of knowledge in the art regarding the maintenance C5 methyltransferase (Dnmt1), there is no information regarding nucleic acid or protein structure and expression or enzymatic properties of the de novo C5 methyltransferase in mammals.